Cryptococcus neoformans and Cryptococcus gattii frequently infect humans to cause life-threatening pneumonia and meningitis resulting in considerable morbidity and mortality in both immunocompromised and immunocompetent hosts. More than one million cases of cryptococcal infection occur globally annually, resulting in >620,000 deaths and up to one-third of all AIDS-associated deaths. An outbreak of C. gattii in the Pacific Northwest involves non-AIDS patients, the majority of whom are immunocompetent, and our investigations document this outbreak has expanded into the US and novel hypervirulent genotypes have emerged. Both Cryptococcus species have a defined sexual cycle involving two opposite mating types (a,a). However, because the vast majority of clinical and environmental isolates are a mating type, it had been unclear if sex occurs in nature and how aerosolized spores thought to be infectious propagules arise. We discovered a novel sexual cycle involving only one mating type, termed a-a same-sex mating or unisexual reproduction. Our population genetic studies provide substantial evidence that unisexual reproduction is a predominant route generating genetic diversity in nature. Our studies further demonstrate that spores produced by sex are infectious following inhalation, and thus unisexual reproduction may be the source of human infections. Candida albicans was also recently discovered to undergo same-sex mating, and cryptic sexual cycles are being found for other fungal and parasite eukaryotic pathogens. Thus, our studies on the roles of sex in pathogen evolution and emergence are of general importance. Our studies supported by this award have defined conditions and genetic circuits governing a-a opposite and a-a unisexual reproduction of Cryptococcus. We have 1) shown that sex occurs in common environmental niches (pigeon guano, plants/trees) and in response to nutrient limitation, light/darkness, carbon dioxide, and inositol; 2) elucidated same-sex genetic cascades involving the novel cell surface receptor Cpr2 and the nuclear transcription factors Mat2 and Znf2; 3) adduced robust population genetic evidence that unisexual reproduction occurs in nature; and 4) shown that sexual reproduction has shaped the C. gattii population structure with implications for pathogen emergence. Our hypothesis is that a-a unisexual reproduction generates genetic diversity and infectious spores. Our recent studies reveal novel features of sexual reproduction allowing us to propose three new aims to test these hypotheses. Aim 1 is to elucidate how the RNAi-dependent sex-induced silencing pathway functions to guard genome integrity. Aim 2 focuses on the de novo generation of genotypic and phenotypic plasticity by sexual reproduction involving aneuploidy and diploidy. Aim 3 addresses sexual reproduction and the basis of hypervirulence in the C. gattii Pacific Northwest outbreak. These studies will advance understanding of how microbial pathogens evolve and emerge via sexual reproduction with implications for treatment and prevention. PUBLIC HEALTH RELEVANCE: These studies focus on the human fungal pathogens Cryptococcus neoformans and Cryptococcus gattii, fungi that infect the lungs and brain to cause life-threatening pneumonia and meningitis. C. neoformans causes >1,000,000 infections annually, and >620,000 deaths and up to one-third of all AIDS related deaths. It is now a greater cause of AIDS related morbidity in Africa than tuberculosis. C. gattii is causing an outbreak in the Pacific Northwest in largely immunocompetent individuals, and has expanded into the United States. Our studies focus on the role of sexual reproduction in Cryptococcus, leading to genetic diversity and the production of infectious spores.